Methods Of Making An Antibody And Compositions Thereof

ABSTRACT

The present invention relates to new methods for making antibodies that involves chemically modifying the antigen with methylglyoxal. The present invention further relates to antibodies made in this manner, and include antibodies specific to LL-37. The present invention pertains to assays that utilize anti-LL-37 antibodies and assess compounds that stimulate LL-37 production. Such compounds are useful in antimicrobial compositions.

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.61/169,845 filed Apr. 16, 2009, entitled “Antibacterial Compositions” byToshihisa Kawai, et al.

The entire teachings of the above application are incorporated herein byreference.

GOVERNMENT SUPPORT

The invention was supported, in whole or in part, by a grant DE-18310from the National Institute of Dental And Craniofacial Research (NIDCR),an institute of National Institute of Health (NIH). The Government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

A protein, known as CAP-18 is a human cathelicidin, which was firstshown to be expressed in certain epithelial cells. The protein ischaracterized by a conserved N-terminal cathelin domain and a variableC-terminal antimicrobial domain. This C-terminal domain can be cleavedoff by proteinases, releasing the active peptide, referred to as LL-37.LL-37 is believed to have antimicrobial activity against variousmicroorganisms.

Compounds that stimulate the antimicrobial activity of LL-37 as well asexpression of LL-37 could be useful in antimicrobial compositions.Antibodies that are specific to CAP-18 and to its active form, LL-37,would be helpful in assessing compounds that stimulate expression ofLL-37.

Accordingly, a need exists to develop antibodies that are specific toCAP-18 and to LL-37. Additionally, a further need exists to developassays to identify compounds that stimulate expression of LL-37 andinduce antimicrobial activity.

SUMMARY OF THE INVENTION

The present invention relates to methods of preparing an immunizingantigen to be used in making an antibody to a native antigen byselecting a native antigen having an amino acid sequence with a highcontent of arginine, lysine, cysteine or a combination thereof; andcontacting the antigen with methylglyoxal in an amount sufficient tochemically modify the antigen to thereby obtain a chemically modifiedantigen; wherein the chemically modified antigen is prepared for use inmaking an antibody specific to the native antigen. In an embodiment, theamino acid sequence includes one having a content of arginine, lysine,cysteine or a combination thereof in a range between about 1% and about30% total content. The present invention includes contacting the antigenwith an amount of methylglyoxal in a range between about 1 nM and about1 mM. Once chemically modified, the present invention includes, e.g.,immunizing an animal with the chemically modified antigen.

In an embodiment, the present invention includes methods of making anantibody to a native antigen by selecting a native antigen having anamino acid sequence with a high content of arginine, lysine, cysteine ora combination thereof; contacting the antigen with methylglyoxal in anamount sufficient to chemically modify the antigen to thereby obtain achemically modified antigen; and injecting the chemically modifiedantigen into an animal in an amount sufficient to illicit an immuneresponse by the animal, wherein the immune response includes theproduction of one or more antibody secreting cells that is specific tothe native antigen; and wherein the antibody to the native antigen ismade. In an aspect, the antibody secreting cell is contacted with amyeloma cell under conditions suitable for fusion thereof to therebyobtain a hybridoma that secretes an antibody specific to the nativeantigen. The steps include, e.g., selecting for hybridomas made duringfusion and isolating the hybridoma that secretes an antibody specific tothe native antigen. Accordingly, the present invention includesantibodies made from the methods described herein.

The present invention also embodies an isolated polypeptide moleculethat includes any one of the following amino acid sequence an amino acidsequence encoded by a nucleic acid molecule having a sequence of SEQ IDNO: 1, 3, or combination thereof; an amino acid sequence encoded by acomplement of SEQ ID NO: 1, 3, or combination thereof; an amino acidsequence encoded by a nucleic acid molecule that hybridizes to SEQ IDNO: 1, 3, or combination thereof; or an amino acid sequence set forth inSEQ ID NO: 2, 4, or combination thereof.

Similarly, the present invention pertains to an isolated nucleic acidmolecule having a nucleic acid sequence including any one of thefollowing: a nucleic acid sequence set forth in SEQ ID NO: 1, 3, orcombination thereof; a nucleic acid sequence that is a complement of SEQID NO: 1, 3, or combination thereof; a nucleic acid sequence thathybridizes to SEQ ID NO:1, 3, or combination thereof; or a nucleic acidsequence that encodes SEQ ID NO: 2, 4, or combination thereof. Theisolated nucleic acid molecule of the present invention can include anucleic acid sequence that encodes a detectable label (e.g.,luciferase). A vector, plasmid or host cell that includes or istransferred with a nucleic acid molecule described herein in alsoencompassed by the present invention. Compositions include thepolypeptide and/or nucleic acid sequences described herein related tothe present invention.

The present invention also pertains to an antibody specific to LL-37,wherein the antibody comprises a variable heavy chain having an aminoacid sequence, as described herein. The antibody can include adetectable label.

Yet in another embodiment, the present invention includes methods formeasuring the presence, absence or amount of LL-37 in a sample bycontacting the sample with an antibody that binds to LL-37 sufficientlyto allow formation of a complex between the sample and the antibody, tothereby form an antigen-antibody complex; and assessing the presence,absence or amount of the antigen-antibody complex. The method, in anaspect, includes comparing the amount of the antigen-antibody complex toa control. In an embodiment, the method includes contacting the samplewith a second antibody specific to LL-37 or said antigen-antibodycomplex. In a particular embodiment, the antibody that binds to LL-37has an amino acid sequence set forth in SEQ ID NO: 1,3, or combinationthereof. The anti-microbial peptide or the antibody can be bound to asolid support.

The present invention further embodies methods for assessing a compoundfor stimulation of LL-37 in a sample by contacting the sample with acompound to be tested to allow stimulation of LL-37; and contacting thesample with an antibody that binds to LL-37 sufficiently to allowformation of a complex between the sample and the antibody, to therebyform an antigen-antibody complex; assessing the presence, absence oramount of the antigen-antibody complex; wherein the presence or anincreased level of the LL-37, as compared to a control, indicates thatthe compound stimulates production of the LL-37, and the absence ordecreased level of LL-37, as compared to a control, indicates that thecompound does not stimulate of the production of the LL-37.

Embodied by the present invention also includes methods for assessing acompound for stimulation of LL-37 in a sample by contacting the samplewith the compound to be tested sufficiently to allow stimulation ofLL-37; contacting the sample with at least two oligonucleotide primersin a polymerase chain reaction, wherein at least one of theoligonucleotide primers is specific for the nucleic acid sequence ofLL-37, sufficiently to allow amplification of the primers; and detectingin the sample the amplified nucleic acid sequence; wherein the presencethe amplified nucleic acid sequence indicates that the compoundstimulates production of the LL-37, and the absence of the amplifiednucleic acid sequence indicates that the compound does not stimulate ofthe production of the LL-37. In an aspect, at least one of theoligonucleotide primers has at least about 10 contiguous bases.

The present invention includes methods for assessing a compound forstimulation of LL-37 in a sample, by contacting the sample with thecompound to be tested sufficiently to allow stimulation of LL-37;contacting the sample with one or more oligonucleotide probes specificfor an isolated nucleic acid molecule of LL-37 under high stringencyconditions, sufficiently to allow hybridization between the sample andthe probe; and detecting the nucleic acid molecule that hybridizes tothe oligonucleotide probe in the sample; wherein the presence thehybridization indicates that the compound stimulates production of theLL-37, and the absence of hybridization indicates that the compound doesnot stimulate the production of the LL-37.

The present invention is advantage for several reasons. The presentinvention provides a new method for increasing antigencity of anantigen, resulting in monoclonal antibodies having higher affinities. Asa result, better assays can be developed. In particular, the presentinvention provides for an LL-37 assay to assess a compounds ability tostimulate anti-microbial activity. This assay provides a way to assesscompounds for potentially new antibacterial medications andcompositions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a sequence listing showing the nucleic acid sequences, SEQ IDNO: 1 and 3, of the hyper-variable region of LL-37 specific heavy chainfragments.

FIG. 2 is a sequence listing showing the amino acid sequences, SEQ IDNO: 2 and 4, of the hyper-variable region of LL-37 specific heavy chainfragments.

FIG. 3 is a graphical representation a standard ELISA curve showing theconcentration of peptide (pg/ml) against OD490 nm.

DETAILED DESCRIPTION OF THE INVENTION

A description of preferred embodiments of the invention follows.

The present invention relates to novel antibody fragments that havespecificity to LL-37, and new methods for making antibodies.Additionally, the present invention relates to assays (e.g., highthroughput assays) using the anti-LL-37 antibodies to assess compoundsthat stimulate LL-37 expression.

Human CAP-18, a human cathelicidin, was first identified in neutrophilsand later shown to be expressed in various squamous epithelia, surfaceepithelial cells of the conducting airways, and serous and mucous cellsof the submucosal glands, by keratinocytes in inflamed skin and byspecific lymphocyte and monocyte populations. Human CAP-18 is the onlyhuman cathelicidin identified to date. It belongs to the cathelicidinfamily of antimicrobial peptides that are characterized by a conservedN-terminal cathelin domain and a variable C-terminal antimicrobialdomain. This C-terminal domain can be cleaved off from the precursor byproteinases, releasing the active peptide. Exocytosed material fromneutrophils contains hCAP-18 that has been proteolytically cleaved byproteinase-3 yielding the 4.5 kD active alpha helical peptide LL-37.LL-37 displays antimicrobial activity against a broad spectrum ofmicroorganisms and possesses synergistic antibacterial effects withother antimicrobial peptides, such as defensins. It is believed thatcathelicidins play a role in effective host defense against infection. Astudy has that mice deficient in the murine cathelicidin-relatedantimicrobial peptide suffer from more severe bacterial skin infections.Tjabringa, Sandra, et al., J. Immunol, 171:6690-6696 (2003). For anotherinstance, deficiency in saliva LL-37 accords with occurrence ofperiodontal disease in patients with morbus Kostmann. Pütsep, Katrin, etal, Lancet, 360:1144-1149 (2002).

As used herein hCAP-18 is considered to be the pro-form of LL-37. Aftercleavage by proteinases, a 4 kD polypeptide results as the active formof LL-37. As used herein, both forms are referred to as LL-37. Theantibodies of the present invention bind to both forms of the LL-37, thepro-form and the active form.

Antibodies specific to LL-37 have often been difficult to obtain and/ormake. Antibodies to the active form of LL-37 have not been able to bedeveloped. LL-37, when injected into mice to make monoclonal antibodies,does not illicit a strong antigen response because LL-37 is highlyconserved between human and animal species, and has little or noMHC-class-II binding epitopes.

It has been determined that LL-37 has antimicrobial properties. Forexample, LL-37 is stimulated by certain compounds such as those havingtrihydroxybenzoate moieties. In particular, trihydroxybenzoatederivatives including 3,4,5-trihydroxybenzoic acid (i.e., gallic acid),its isoform, 2,3,4-trihydroxybenzoic acid, C₁₋₄alkyl3,4,5-trihydroxybenzoate (e.g., ethyl 3,4,5-trihydroxybenzoate),C₁₋₄alkyl 2,3,4-trihydroxybenzoate (e.g., ethyl2,3,4-trihydroxybenzoate), EGCG and mixtures thereof. In particular,“THBC” refers to chemical structures such as trihydroxybenzoates thatcomprise tri-hydroxy benzoic acid and its isoforms, which are found inthe polyphenol extracts of natural plant products or in antioxidant foodpreservatives. As such trihydroxybenzoate moieties can be used as apositive control the LL-37 assay described herein.

Compounds for stimulating production of anti-microbial peptide LL-37 canbe helpful in treating bacterial infection. Particularly, suchcompositions are particularly useful for treating a bacterial infectionwherein (a) the bacteria is antibiotic resistant, (b) the patient isallergic to antibiotics; (c) the patient is immuno-compromised (e.g.,HIV/AIDS patients); or (d) the infection is of an oral cavity.Additionally, LL-37 is a potent antimicrobial against variousstaphylococcal species and particularly virulent antibiotic-resistantstrains of pathogenic bacteria, such as those found inMethicilin-resistant Staphylococcus aureus (MRSA). Such compounds canalso be useful for 1) oral health care products, 2) skin and mucosalhealth care products, 3) antimicrobial drugs, and 4) neutraceuticalproducts.

Antibodies for Assessing LL-37:

Antibodies that are specific to LL-37 are helpful in determiningcompounds that stimulate LL-37 production, and would be helpful intreating the above disease or conditions. Compounds that stimulate LL-37production can be included in compositions in which anti-microbialproperties are needed. Since it is also reported that LL-37 can downregulate the production of pro-inflammatory cytokines, the increasedproduction of LL-37 also results in the suppression of inflammatoryresponses. Scott M G, J Immunol. 2002; 169:3883-389 (2002). Compoundsthat stimulate LL-37 can be included in health compositions (e.g.,toothpaste, mouth wash, floss) and medications to treat various disease,including those related to the gut (e.g., for Crohn's disease orColitis), and the like.

Accordingly, a need exists for antibodies that are specific for LL-37 toscreen for compounds that stimulate LL-37 production. A need exists fora method of making antibodies specific to LL-37, and other antigens forwhich antibodies are difficult to obtain.

The present invention relates to an antibody specific to LL-37. Asdescribed in the exemplification section, two antibodies were made andisolated, and are referred to as L2, made by hybridoma cell line calledAL37-2, and L7, made by hybridoma cell line called AL37-7.

The term, “antibody,” encompasses polyclonal antibodies, monoclonalantibodies, single chain antibodies, VHH antibodies, chimeric,humanized, primatized, CDR-grafted, and veneered antibodies. This termfurther includes portions derived from different species, humanantibodies which are native or derived from combinatorial libraries, andthe like. Conventional techniques can chemically join together thevarious portions of these antibodies. Genetic engineering techniques canalso prepare the antibody as a contiguous protein. For example, nucleicacids encoding a chimeric or humanized chain can be expressed to producea contiguous protein.

Specifically, the term “antibody fragment” refers to portion of animmunoglobulin having specificity to the LL-37 or a portion thereof. Theterm, “antibody fragment”, is intended to encompass fragments from bothpolyclonal and monoclonal antibodies including transgenically producedantibodies, single-chain antibodies (scFvs), recombinant Fabs, andrecombinant camelid heavy-chain-only antibodies (VHHs). VHHs are alsoreferred to as nanobodies.

Functional fragments of antibodies, including fragments of chimeric,humanized, primatized, veneered or single chain antibodies, can also beproduced. Functional fragments or portions of the foregoing antibodiesinclude those which are reactive with the LL-37. For example, antibodyfragments capable of binding to LL-37 or portion thereof, including, butnot limited to scFvs, Fabs, VHHs, Fv, Fab, Fab′ and F(ab′)₂ areencompassed by the invention. Such fragments can be produced byenzymatic cleavage or by recombinant techniques. For instance, papain orpepsin cleavage can generate Fab or F(ab′)2 fragments, respectively.Antibodies can also be produced in a variety of truncated forms usingantibody genes in which one or more stop codons has been introducedupstream of the natural stop site. For example, a chimeric gene encodinga F(ab′)₂ heavy chain portion can be designed to include DNA sequencesencoding the CH1 domain and hinge region of the heavy chain.Accordingly, the present invention encompasses a polynucleic acid thatencodes the anti-LL-37 antibody fragments including heavy chain variableregions, which can optionally further nucleic acid that encodes a tag ordetector (e.g., luciferase report gene).

The term “antibody” also includes various forms of modified antibodies.For example, modification may occur by directly or indirectly attachinga detectable label. The detectable labels may include a radioisotope,spin label, antigen label such as a FLAG tag, enzyme label, fluorescentor chemiluminescent group and the like.

The term “sample” includes fluid and/or cells from an oral swab (e.g.,containing saliva and/or epithelial cells from the oral cavity), tissue,fluid, whole blood, plasma, serum and aqueous blood components from apatient.

Methods of Assessment of LL-37 and Methods of Making an Anti-LL-37Antibody:

Method for assessing the presence, absence or level of LL-37, in asample, is encompassed by the present invention. Suitable assays includeimmunological methods, such as high throughput assays, radioimmunoassay,enzyme-linked immunosorbent assays (ELISA), chemiluminescence assays,and rapid immunochromatographic assays. A high throughput assays is apreferred embodiment of the present invention. Any method known now ordeveloped later can be used for measuring LL-37 using the antibodiesdescribed herein.

The antibodies described herein are monoclonal antibodies. However, oneor more variable regions of the monoclonal antibody can be constructedinto a synthetic antibody having an antibody backbone, or variablefragments of the antibody can be used to detect LL-37 expression. Singlechain antibody using one or more variable regions can be used.Accordingly, the term antibody is intended to encompass monoclonalantibodies, and functional fragments thereof.

In several of the preferred embodiments, immunological techniques detectthe presence, absence, or levels of LL-37 described herein by means ofan anti-LL-37 antibody (i.e., one or more antibodies). The term“anti-LL-37” includes one or more monoclonal antibodies or fragmentsthereof, and mixtures or cocktails thereof, and refers to antibodiesspecific to polypeptides having a sequence set forth in SEQ ID Nos: 2,4, or combination thereof, or portions thereof.

Anti-LL-37 antibodies can be raised against appropriate immunogens, suchas isolated and/or recombinant LL-37 polypeptides described herein,analogs or portion thereof (including synthetic molecules, such assynthetic peptides). Developing an antigenic response in the mice andobtaining high affinity antibodies to LL-37 was a challenge. Conservedamino acid sequences of LL-37 between human and animals in the range ofabout 70% to about 80% exist, and so mice injected with LL-37, even withan adjuvant, did not illicit a significant antigenic response thatallowed one to obtain an specific and high affinity LL-37 antibody.Additionally, LL-37 has little or no MHC-class-II binding epitopes inits sequence, and therefore, does not easily allow absorption by theantigen presenting cells, making it more difficult to obtain the desiredantigenic response in the animal. No MHC-class-II binding motif in LL-37peptide was detected by RankPep algorism that evaluates the peptidebinding capacity to mouse MHC-class-II [mouse strains testes, BALB/c andC57BL/6]: http://bio.dfci.harvard.edu/RANKPEP/.

To increase immunogenicity, the LL-37 protein underwent a chemicalmodification prior to being injected into an animal (e.g., mouse,rodent, rabbit, goat, monkey, camel, and the like) during the monoclonalantibody procedures. The chemical modification allows for a reactionwith certain amino acids present in the protein. In this case,methylglyoxal was used to react with Arginine (R) and Lysine (K) presentin LL-37. When methylglyoxal was combined with LL-37, the absorption byantigen presenting cells increased to thereby increase immunogenicityand develop a LL-37 specific antibody having a high affinity. Antigenpresenting cells express the specific receptor (RAGE; the receptor foradvanced glycation end products) for methylglyoxal-modified peptide andthereby allow for better absorption.

Chemical modification of proteins prior to injection into an animal canbe applied to proteins other than LL-37 for the production of monoclonalantibodies. In particular, any protein having arginine (R), lysine (K)or cysteine (C) can be chemically modified with methylglyoxal forpurposes of increasing an antigenic response.

Methylglyoxal is contacted with the antigen to be modified in an amountbetween about 1 nM and about 1 mM. See Example 1.

Advanced Glycation End products (AGEs) are the result of nonenzymaticglycation of proteins and lipids, as a consequence of a chain ofchemical reactions after an initial glycation reaction. Many cells inthe body, especially immune cells express the Receptor for AdvancedGlycation End products (RAGE) that, when binding AGEs, contributes toimmune and inflammatory responses. Recent research has shown thatphysiological glycation processes also involve the formation of advancedglycation endproducts (AGEs) by reactive alpha-oxoaldehydes particularlyglyoxal, methylglyoxal and 3-deoxyglucosone (3-DG), and others.

Accordingly, a native antigen can be chemically modified, as describedherein, by AGEs including by reactive alpha-oxoaldehydes particularlyglyoxal, methylglyoxal and 3-deoxyglucosone (3-DG),

In an embodiment, the amino acid content of the protein to be chemicallymodified includes at least about 5% arginine (R), lysine (K), cysteine(C), or combination of both of its total amino acid content (e.g., about1%, 5%, 10%, 15%, 20%, 25%, 30% or 40%). The LL-37 protein is abundantin both arginine and lysine, and specifically has about 5 arginines and6 lysines, while lacking cyctein among a total 37 amino acids. The fullamino acid sequence of LL-37 is LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES(SEQ ID NO: 5). Using this method, two MAbs were generated and bind athigh affinity to different epitopes present in LL-37. Chemicallymodifying LL-37 to increase antigencity allowed mAbs specific to theactive form of LL-37, in addition to the pro-form of LL-37.

In one embodiment, antibodies are raised against an isolated,recombinant, active, chemically modified LL-37 polypeptide describedherein or portion thereof (e.g., a peptide) or against a host cell whichexpresses recombinant or chemically modified LL-37. In addition, cellsexpressing recombinant or modified antigenic LL-37 polypeptidesdescribed herein, such as transfected cells, can be used as immunogensor in a screen for antibody which binds receptor.

Preparing the immunizing antigen can be done, as described above and anysuitable technique, now known or later developed, can be used to producepolyclonal or monoclonal antibodies. The art contains a variety of thesemethods (see e.g., Kohler et al., Nature, 256: 495-497 (1975) and Eur.J. Immunol. 6: 511-519 (1976); Milstein et al., Nature 266: 550-552(1977); Koprowski et al., U.S. Pat. No. 4,172,124; Harlow, E. and D.Lane, 1988, Antibodies: A Laboratory Manual, (Cold Spring HarborLaboratory: Cold Spring Harbor, N.Y.); Current Protocols In MolecularBiology, Vol. 2 (Supplement 27, Summer '94), Ausubel, F. M. et al.,Eds., (John Wiley & Sons: New York, N.Y.), Chapter 11, (1991)).Generally, fusing a suitable immortal or myeloma cell line, such asSP2/0, with antibody producing cells can produce a hybridoma. Animalsimmunized with the antigen of interest, after chemical modification,provide a cell that produces an antibody. Surprisingly, one or moreantibodies specific for the native antigen are made and have a highbinding affinity to the native antigen, even though the immunizingantigen was chemically modified. The immunized animal generally producescells that make antibody that is specific to the antigen and these cellsare typically cells from the spleen or lymph nodes. Cells from thespleen or lymph node are teased or separated from one another andcultured. Polyethylene glycol (PEG) or similar compound is used to fusethe myeloma cell line with the spleen or lymph node cells and aselective medium is used in which only fused cells can grow. Selectiveculture conditions isolate antibody producing hybridoma cells whilelimiting dilution techniques produce them. This mixture of cells isdiluted and clones are grown from single parent cells generally in wellsof a plate. The antibodies secreted by the different clones are thenassayed for their ability to bind to the antigen. Researchers can usesuitable assays such as ELISA to select antibody producing cells withthe desired specificity. The hybridomas can be grown indefinitely in asuitable cell culture media, or they can be injected in mice into theperitoneal cavity, wherein they produce tumors containing anantibody-rich fluid called ascites fluid. The ascites that contains theantibody can be drained, bled or otherwise withdrawn from the animal.The antibody can be purified using standard purification techniques(e.g., ultra-filtration, dialysis, and chromatography).

Other suitable methods can produce or isolate antibodies of therequisite specificity. Examples of other methods include selectingrecombinant antibody from a library or relying upon immunization oftransgenic animals such as mice to make human monoclonal antibodies.

According to the method, an assay can determine the presence, absence orlevel of LL-37 peptides in a biological sample. Such an assay includescombining the sample to be tested with an antibody having specificityfor LL-37 described herein, under conditions suitable for formation of acomplex between antibody and LL-37, and detecting or measuring (directlyor indirectly) the formation of a complex. In the event that a compoundis being assessed, the sample can be combined with the compound to betested and incubated under conditions to allow for expression of LL-37.The sample can be obtained directly or indirectly (e.g., provided by ahealthcare provider), and can be prepared by a method suitable for theparticular sample (e.g., saliva, epithelial cells from the oral cavity,urine, sputum, fecal matter, cerebral spinal fluid, whole blood,platelet rich plasma, platelet poor plasma, serum) and select an assayformat. Methods of combining sample and antibody, and methods ofdetecting complex formation are also selected to be compatible with theassay format.

The assays described herein can be modified to assess the effect of acompound on LL-37 expression. In such a case, the compound can beintroduced to the assay by contacting the compound with cells or samplesthat can express LL-37. The cells or sample can be subjected to compoundto be tested to determine if the compound has an effect on LL-37expression. Alternatively, the subject, from whom the sample is taken,can be subjected to the compound and then a sample is taken. The samplecan then be assessed for LL-37 expression, as described herein. Since ithas been determined that trihydroxybenzoate stimulates LL-37 expression,a trihydroxybenzoate moiety can be used as a positive control in theassay.

Suitable labels can be detected directly, such as radioactive,fluorescent or chemiluminescent labels. They can also be indirectlydetected using labels such as enzyme labels and other antigenic orspecific binding partners like biotin. Examples of such labels includefluorescent labels such as fluorescein, rhodamine, chemiluminescentlabels such as luciferase, radioisotope labels such as 32P, 125I, 131I,enzyme labels such as horseradish peroxidase, and alkaline phosphatase,galactosidase, biotin, avidin, spin labels and the like. The detectionof antibodies in a complex can also be done immunologically with asecond antibody, which is then detected (e.g., by means of a label).Conventional methods or other suitable methods can directly orindirectly label an antibody. Labeled primary and secondary antibodiescan be obtained commercially or prepared using methods know to one ofskill in the art (see Harlow, E. and D. Lane, 1988, Antibodies: ALaboratory Manual, (Cold Spring Harbor Laboratory: Cold Spring Harbor,N.Y.).

In a preferred embodiment, the presence, absence, or level of LL-37 in asample is determined using a high throughput assay, an ELISA assay, asandwich ELISA assay, or immunochromatographic assay.

For detection of LL-37 in a suitable sample, a sample (e.g., saliva) iscollected. Samples can be processed as known in the art. The compound tobe assessed can be contacted with the sample, or in contact with thesubject prior to taking the sample. For example, the compound can bepart of an oral composition that the subject uses.

In an embodiment, LL-37 is determined using an ELISA assay or a sandwichELISA assay. In one embodiment, murine L2 is used as capture antibodyand murine L7 is used as detector antibody.

In an embodiment, the assay further includes combining a suitablesample, and optionally the compound to be tested, with a compositionhaving an anti-LL-37 polypeptide antibody as detector (e.g.,biotinylated anti-LL-37 polypeptides MAb and HRP-streptavidin, orHRP-conjugated anti-LL-37 polypeptides Mab), and a solid support, suchas a microtiter plate, having an anti-LL-37 polypeptide capture antibodybound (directly or indirectly) thereto. The detector antibody binds to adifferent LL-37 polypeptide epitope from that recognized by the captureantibody, under conditions suitable for the formation of the complex.The assay then involves determining the formation of complex in thesamples. The presence of or increase in LL-37 in a sample of anindividual indicates the presence of a compound that increasedexpression or production of LL-37, whereas the absence of or decrease ina LL-37 polypeptide indicates the compound to be tested that does notincrease expression or production of LL-37.

The solid support, such as a microtiter plate, dipstick, bead, pad,strip, or other suitable support, can be coated directly or indirectlywith an anti-LL-37 polypeptide antibody or LL-37 specific antigen. Forexample, an anti-LL-37 polypeptide antibody can coat a microtiter well,or a biotinylated anti-LL-37 polypeptide Mab can be added to astreptavidin coated support. With respect to a immunochromatographicassay, a pad or strip can be coated with an antibody specific for theantigen, and when a sample having the one or more of antigens describedherein comes into contact with the antibody, the complex can turn acolor with aid of a detector, as further described herein. A variety ofimmobilizing or coating methods as well as a number of solid supportscan be used, and can be selected according to the desired format.

In another embodiment, the sample (or an LL-37 polypeptide standard) iscombined with the solid support simultaneously with the detectorantibody, and optionally with a one or more reagents by which detectionis monitored. For example, the sample can be combined with the solidsupport simultaneously with (a) HRP-conjugated anti-LL-37 polypeptideMab, or (b) a biotinylated anti-LL-37 polypeptide Mab andHRP-streptavidin.

A known amount of an LL-37 polypeptide standard can be prepared andprocessed as described above for a suitable sample. This LL-37polypeptide standard assists in quantifying the amount of LL-37 detectedby comparing the level of LL-37 in the sample relative to that in thestandard. In one embodiment, active LL-37 is used as a standard.

A physician, technician, apparatus or a qualified person can compare theamount of detected complex with a suitable control to determine if theLL-37 levels are elevated or not. A control can be the level of LL-37 ina sample take from the subject, but not subjected to the compound to betested. A positive control can be the level of LL-37 subjected to aknown compound that stimulates LL-37 express (e.g., trihydroxybenzoatemoiety, or EGCG). A control can also be the average level of LL-37 forthe particular sample, in a healthy population.

Typical assays for LL-37 are sequential assays in which a plate iscoated with first antibody, sample is added, the plate is washed, secondtagged antibody is added, and the plate is washed and bound secondantibody is quantified. In another embodiment, a format in whichantibodies and the sample are added simultaneously, in a competitiveELISA format, can achieve greater sensitivity.

A variety of methods can determine the amount of LL-37 in complexes. Forexample, when HRP is used as a label, a suitable substrate such as OPDcan be added to produce color intensity directly proportional to thebound anti-LL-37 polypeptides mAb (assessed e.g., by optical density),and therefore to the LL-37 in the sample.

A technician, physician, qualified person or apparatus can compare theresults to a suitable control such as a standard, or baseline levels ofLL-37 in a sample from the same donor. For example, the assay can beperformed using a known amount of LL-37 standard in lieu of a sample,and a standard curved established. One can relatively compare knownamounts of the LL-37 standard to the amount of complex formed ordetected.

The Anti-LL-37 Antibody Polypeptides and their Function

The present invention relates to isolated polypeptide moleculesincluding anti-LL-37 antibodies or portions thereof. The presentinvention includes polypeptide molecules that contain any one of theanti-LL-37 antibody amino acid sequences (SEQ ID NO: 2, 4, orcombinations thereof). The present invention also pertains topolypeptide molecules that are encoded by nucleic acid sequences, SEQ IDNO: 1, 3, or combinations thereof).

As used herein, the term “polypeptide” encompasses amino acid chains ofany length (e.g., heavy chains of variable regions), including fulllength proteins, wherein the amino acid residues are linked by covalentpeptide bonds. Thus, a polypeptide comprising a heavy chain of any ofthe sequences can consist entirely of the heavy chain, or can containadditional sequences. The additional sequences can be derived from theantibody backbones, or can be heterologous. In general, the polypeptidesdisclosed herein are prepared such that they are in substantially pureform. Preferably, the polypeptides are at least about 80% pure, morepreferably at least about 90% pure and most preferably at least about99% pure.

Polypeptides of the present invention referred to herein as “isolated”are polypeptides that separated away from other proteins and cellularmaterial of their source of origin. Anti-LL-37 antibody polypeptidesinclude peptides derived by the monoclonal/polyclonal process describedherein, essentially pure protein, proteins produced by chemicalsynthesis, by combinations of biological and chemical synthesis and byrecombinant methods. The proteins of the present invention have beenisolated and characterized as to its physical characteristics using theprocedures described herein and in the Exemplification. Such techniquesinclude, for example, the monoclonal/polyclonal antibody making process,salting out, immunoprecipation, column chromatography, high pressureliquid chromatography or electrophoresis.

The compositions and methods of the present invention also encompassvariants of the above polypeptides and DNA molecules. A polypeptide“variant,” as used herein, is a polypeptide that differs from therecited polypeptide only in conservative substitutions and/ormodifications, such that the polypeptide binding and/or affinitypertaining to LL-37 properties (e.g., specificity, affinity, orcombination thereof) of the polypeptide are retained. Polypeptidevariants preferably exhibit at least about 60%, more preferably at leastabout 70% and most preferably at least about 80% homology to theidentified polypeptides (e.g., about 60%, 65%, 70%, 75%, 80%, 85%, 90%,95% homology). For polypeptides with LL-37 binding and/or affinityproperties, variants can, alternatively, be identified by modifying theamino acid sequence of one of the above polypeptides, and evaluating thebinding and/or affinity of the modified polypeptide. Such modifiedsequences can be prepared and tested using, for example, therepresentative procedures described herein (e.g., an ELISA). Thehomology between the two isolated antibody sequences is about 63%. Mostof homologous sequence is derived from the frame work of hypervariableregion of immunoglobulin. Please see below).

ClustalW sequence alignment result 37-2VAPRLWHLSQLWMHWVKQRPGHGLEWIGEIDPSDSDTNYNQKFKGKATLTVDKSSSTAYM  60SEQ ID NO: 1 37-7-----SGLDILILTMVVLTT-------------------TQKFKGKATLTVDKSSSTAYM 36SEQ ID NO: 3       *. * :  *   .                   .******************** 37-2QLSSLTSEDSAVYYCAKTQ  79 37-7 XLSSLTSEDSAVYYCARHM  55  ***************:

The polypeptides of the present invention, including a full lengthsequence, partial sequences, functional fragments and homologues, thatallow for or assist in binding to or having affinity for LL-37.“Binding”, as used herein, refers to the ability to for the antibody toattach to or otherwise be specific for portion of LL-37, e.g., throughhydrogen bonds. “Affinity” refers to the degree to which the antibody ofthe present invention binds to LL-37.

As used herein, a “conservative substitution” is one in which an aminoacid is substituted for another amino acid that has similar properties,such that one skilled in the art of peptide chemistry would expect thesecondary structure and hydropathic nature of the polypeptide to besubstantially unchanged. In general, the following groups of amino acidsrepresent conservative changes: (1) ala, pro, gly, glu, asp, gln, asn,ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4)lys, arg, his; and (5) phe, tyr, trp, his.

Variants can also, or alternatively, contain other modifications,including the deletion or addition of amino acids that have minimalinfluence on the antigenic properties, secondary structure andhydropathic nature of the polypeptide. For example, a polypeptide can beconjugated to a signal (or leader) sequence at the N-terminal end of theprotein which co-translationally or post-translationally directstransfer of the protein. The polypeptide can also be conjugated to alinker or other sequence for ease of synthesis, purification oridentification of the polypeptide (e.g., poly-His), or to enhancebinding of the polypeptide to a solid support. For example, apolypeptide can be conjugated to an immunoglobulin Fc region.

The present invention also encompasses anti-LL-37 antibodies, chainsthereof, polypeptides, variants thereof, or those having amino acidsequences analogous to the amino acid sequences of anti-LL-37 antibodiesdescribed herein. Such polypeptides are defined herein as anti-LL-37antibody analogs (e.g., homologues), or mutants or derivatives.“Analogous” or “homologous” amino acid sequences refer to amino acidsequences with sufficient identity of any one of the anti-LL-37 antibodysequences so as to possess the biological activity (e.g., the ability tobind to LL-37 and/or have affinity for LL-37) of any one of the nativeanti-LL-37 antibodies. For example, an analog polypeptide can beproduced with “silent” changes in the amino acid sequence wherein one,or more, amino acid residues differ from the amino acid residues of anyone of the anti-LL-37 antibodies, yet still possess the function orbiological activity of the anti-LL-37 antibodies. Examples of suchdifferences include additions, deletions or substitutions of residues ofthe amino acid sequence of anti-LL-37 antibodies described herein. Alsoencompassed by the present invention are analogous polypeptides thatexhibit greater, or lesser, biological activity of any one of theanti-LL-37 antibodies of the present invention. Such polypeptides can bemade by mutating (e.g., substituting, deleting or adding) one or moreamino acid or nucleic acid residues to any of the isolated anti-LL-37antibodies described herein. Such mutations can be performed usingmethods described herein and those known in the art. In particular, thepresent invention relates to homologous polypeptide molecules having atleast about 70% (e.g., 75%, 80%, 85%, 90% or 95%) identity or similaritywith SEQ ID NO: 2, 4, or combination thereof. Percent “identity” refersto the amount of identical nucleotides or amino acids between twonucleotides or amino acid sequences, respectfully. As used herein,“percent similarity” refers to the amount of similar or conservativeamino acids between two amino acid sequences.

Homologous polypeptides can be determined using methods known to thoseof skill in the art. Initial homology searches can be performed at NCBIagainst the GenBank, EMBL and SwissProt databases using, for example,the BLAST network service. Altschuler, S. F., et al., J. Mol. Biol.,215:403 (1990), Altschuler, S. F., Nucleic Acids Res., 25:3389-3402(1998). Computer analysis of nucleotide sequences can be performed usingthe MOTIFS and the FindPatterns subroutines of the Genetics ComputingGroup (GCG, version 8.0) software. Protein and/or nucleotide comparisonswere performed according to Higgins and Sharp (Higgins, D. G. and Sharp,P. M., Gene, 73:237-244 (1988) e.g., using default parameters).

Additionally, the individual isolated polypeptides of the presentinvention are biologically active or functional fragments (e.g.,fragments of heavy chains). The present invention includes fragments ofthese isolated amino acid sequences, yet possesses the function orbiological activity of the sequence. For example, polypeptide fragmentscomprising variable regions of the anti-LL-37 antibodies can be designedand expressed by well-known laboratory methods. Fragments, homologues,or analogous polypeptides can be evaluated for biological activity, asdescribed herein.

The present invention also encompasses biologically active derivativesor analogs of the above described anti-LL-37 antibodies, referred toherein as peptide mimetics. Mimetics can be designed and produced bytechniques known to those of skill in the art. (see e.g., U.S. Pat. Nos.4,612,132; 5,643,873 and 5,654,276). These mimetics can be based, forexample, on an anti-LL-37 antibody amino acid sequence and maintain therelative position in space of the corresponding amino acid sequence.These peptide mimetics possess biological activity similar to thebiological activity of the corresponding peptide compound, but possess a“biological advantage” over the corresponding anti-LL-37 antibody aminoacid sequence with respect to one, or more, of the following properties:solubility, stability and susceptibility to hydrolysis and proteolysis.

Methods for preparing peptide mimetics include modifying the N-terminalamino group, the C-terminal carboxyl group, and/or changing one or moreof the amino linkages in the peptide to a non-amino linkage. Two or moresuch modifications can be coupled in one peptide mimetic molecule.Modifications of peptides to produce peptide mimetics are described inU.S. Pat. Nos. 5,643,873 and 5,654,276. Other forms of the anti-LL-37antibodies, encompassed by the present invention, include those whichare “functionally equivalent.” This term, as used herein, refers to anynucleic acid sequence and its encoded amino acid, which mimics thebiological activity of the anti-LL-37 antibody and/or functional domainsthereof.

Anti-LL-37 antibody Sequences, Plasmids, Vectors and Host Cells

The present invention, in one embodiment, includes an isolated nucleicacid molecule having a sequence of SEQ ID NO: 1, 3, or combinationsthereof. The present invention includes sequences of the variable heavychain, as recited in FIGS. 8 and 9.

As used herein, the terms “DNA molecule” or “nucleic acid molecule”include both sense and anti-sense strands, cDNA, genomic DNA,recombinant DNA, RNA, and wholly or partially synthesized nucleic acidmolecules. A nucleotide “variant” is a sequence that differs from therecited nucleotide sequence in having one or more nucleotide deletions,substitutions or additions. Such modifications can be readily introducedusing standard mutagenesis techniques, such as oligonucleotide-directedsite-specific mutagenesis as taught, for example, by Adelman et al. (DNA2:183, 1983). Nucleotide variants can be naturally occurring allelicvariants, or non-naturally occurring variants. Variant nucleotidesequences preferably exhibit at least about 70%, more preferably atleast about 80% and most preferably at least about 90% homology to therecited sequence. Such variant nucleotide sequences will generallyhybridize to the recited nucleotide sequence under stringent conditions.In one embodiment, “stringent conditions” refers to prewashing in asolution of 6×SSC, 0.2% SDS; hybridizing at 65° Celsius, 6×SSC, 0.2% SDSovernight; followed by two washes of 30 minutes each in 1×SSC, 0.1% SDSat 65° C. and two washes of 30 minutes each in 0.2×SSC, 0.1% SDS at 65°C.

The present invention also encompasses isolated nucleic acid sequencesthat encode anti-LL-37 antibodies, and in particular, those which encodea polypeptide molecule having an amino acid sequence of SEQ ID NO: 2, 4,or combinations thereof. These nucleic acid sequences encodepolypeptides that bind LL-37 and/or are involved the functions furtherdescribed herein.

As used herein, an “isolated” gene or nucleotide sequence which is notflanked by nucleotide sequences which normally (e.g., in nature) flankthe gene or nucleotide sequence (e.g., as in genomic sequences) and/orhas been completely or partially purified from other transcribedsequences (e.g., as in a cDNA or RNA library). Thus, an isolated gene ornucleotide sequence can include a gene or nucleotide sequence which issynthesized chemically or by recombinant means. Nucleic acid constructscontained in a vector are included in the definition of “isolated” asused herein. Also, isolated nucleotide sequences include recombinantnucleic acid molecules and heterologous host cells, as well as partiallyor substantially or purified nucleic acid molecules in solution. In vivoand in vitro RNA transcripts of the present invention are alsoencompassed by “isolated” nucleotide sequences.

The nucleic acid sequences of the present invention include homologuesnucleic acid sequences. “Analogous” or “homologous” nucleic acidsequences refer to nucleic acid sequences with sufficient identity ofany one of the anti-LL-37 antibody nucleic acid sequences, such thatonce encoded into polypeptides, they possess the biological activity ofany one of the anti-LL-37 antibodies described herein. For example, ananalogous nucleic acid molecule can be produced with “silent” changes inthe sequence wherein one, or more, nucleotides differ from thenucleotides of any one of the anti-LL-37 antibodies described herein,yet, once encoded into a polypeptide, still possesses its function orbiological activity. Examples of such differences include additions,deletions or substitutions. Also encompassed by the present inventionare nucleic acid sequences that encode analogous polypeptides thatexhibit greater, or lesser, biological activity of the anti-LL-37antibodies of the present invention. In particular, the presentinvention is directed to nucleic acid molecules having at least about70% (e.g., 75%, 80%, 85%, 90% or 95%) identity with SEQ ID NO: 1, 3, orcombinations thereof.

The nucleic acid molecules of the present invention, including the fulllength sequences, the partial sequences, functional fragments andhomologues, once encoded into polypeptides, bind to LL-37, or has thefunction of the polypeptide, as further described herein. The homologousnucleic acid sequences can be determined using methods known to those ofskill in the art, and by methods described herein including thosedescribed for determining homologous polypeptide sequences. Immunogenicantigens can then be sequenced using techniques such as Edman chemistry.See Edman and Berg, Eur. J. Biochem. 80:116-132, 1967.

Also encompassed by the present invention are nucleic acid sequences,DNA or RNA, which are substantially complementary to the DNA sequencesencoding the anti-LL-37 antibodies of the present invention, and whichspecifically hybridize with their DNA sequences under conditions ofstringency known to those of skill in the art. As defined herein,substantially complementary means that the nucleic acid need not reflectthe exact sequence of the anti-LL-37 antibody sequences, but must besufficiently similar in sequence to permit hybridization with nucleicacid sequence under high stringency conditions. For example,non-complementary bases can be interspersed in a nucleotide sequence, orthe sequences can be longer or shorter than the nucleic acid sequence,provided that the sequence has a sufficient number of basescomplementary to the sequence to allow hybridization therewith.Conditions for stringency are described in e.g., Ausubel, F. M., et al.,Current Protocols in Molecular Biology, (Current Protocol, 1994), andBrown, et al., Nature, 366:575 (1993); and further defined inconjunction with certain assays.

Also encompassed by the present invention are nucleic acid sequences,genomic DNA, cDNA, RNA or a combination thereof, which are substantiallycomplementary to the DNA sequences of the present invention and whichspecifically hybridize with the anti-LL-37 antibody nucleic acidsequences under conditions of sufficient stringency (e.g., highstringency) to identify DNA sequences with substantial nucleic acididentity.

The present invention also includes portions and other variants ofanti-LL-37 antibodies that are generated by synthetic or recombinantmeans. Synthetic polypeptides having fewer than about 100 amino acids,and generally fewer than about 50 amino acids, can be generated usingtechniques well known to those of ordinary skill in the art. Forexample, such polypeptides can be synthesized using any of thecommercially available solid-phase techniques, such as the Merrifieldsolid-phase synthesis method, where amino acids are sequentially addedto a growing amino acid chain. See Merrifield, J. Am. Chem. Soc.85:2149-2146, 1963. Equipment for automated synthesis of polypeptides iscommercially available from suppliers such as Applied BioSystems, Inc.,Foster City, Calif., and can be operated according to the manufacturer'sinstructions. Variants of a native antigen can generally be preparedusing standard mutagenesis techniques, such as oligonucleotide-directedsite-specific mutagenesis. Sections of the DNA sequence can also beremoved using standard techniques to permit preparation of truncatedpolypeptides.

In another embodiment, the present invention includes nucleic acidmolecules (e.g., probes or primers) that hybridize to the anti-LL-37antibody sequences, SEQ ID NO:1, 3, or combinations thereof under highor moderate stringency conditions. In one aspect, the present inventionincludes molecules that are or hybridize to at least about 20 contiguousnucleotides or longer in length (e.g., 50, 100, 200, 300, 400, 500, 600,700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800,1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000,3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, or 4000). Suchmolecules hybridize to one of anti-LL-37 antibody nucleic acid sequencesunder high stringency conditions. The present invention includes suchmolecules and those that encode a polypeptide that has the functions orbiological activity described herein.

Typically the nucleic acid probe comprises a nucleic acid sequence (e.g.SEQ ID NO: 1, 3, or combinations thereof) and is of sufficient lengthand complementarity to specifically hybridize to a nucleic acid sequencethat encodes anti-LL-37 polypeptide. For example, a nucleic acid probecan be at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%the length of the anti-LL-37 antibody nucleic acid sequence. Therequirements of sufficient length and complementarity can be easilydetermined by one of skill in the art. Suitable hybridization conditions(e.g., high stringency conditions) are also described herein.Additionally, the present invention encompasses fragments of thepolypeptides of the present invention or nucleic acid sequences thatencodes a polypeptide wherein the polypeptide has the biologicallyactivity of the anti-LL-37 polypeptides recited herein.

Such fragments are useful in making polypeptide fragments that encodethe variable region of the anti-LL-37 antibody. Antibody fragments thatbind LL-37 are useful in blocking or inhibiting LL-37 function. They arealso useful in performing assays to detect or determine levels of LL-37.With such fragments, compounds that affect LL-37 activity can beassayed. For example, nucleic acid fragments which encode any one of thedomains described herein are also implicated by the present invention.

Stringency conditions for hybridization refers to conditions oftemperature and buffer composition which permit hybridization of a firstnucleic acid sequence to a second nucleic acid sequence, wherein theconditions determine the degree of identity between those sequenceswhich hybridize to each other. Therefore, “high stringency conditions”are those conditions wherein only nucleic acid sequences which are verysimilar to each other will hybridize. The sequences can be less similarto each other if they hybridize under moderate stringency conditions.Still less similarity is needed for two sequences to hybridize under lowstringency conditions. By varying the hybridization conditions from astringency level at which no hybridization occurs, to a level at whichhybridization is first observed, conditions can be determined at which agiven sequence will hybridize to those sequences that are most similarto it. The precise conditions determining the stringency of a particularhybridization include not only the ionic strength, temperature, and theconcentration of destabilizing agents such as formamide, but alsofactors such as the length of the nucleic acid sequences, their basecomposition, the percent of mismatched base pairs between the twosequences, and the frequency of occurrence of subsets of the sequences(e.g., small stretches of repeats) within other non-identical sequences.Washing is the step in which conditions are set so as to determine aminimum level of similarity between the sequences hybridizing with eachother. Generally, from the lowest temperature at which only homologoushybridization occurs, a 1% mismatch between two sequences results in a1° C. decrease in the melting temperature (Tm) for any chosen SSCconcentration. Generally, a doubling of the concentration of SSC resultsin an increase in the Tm of about 17° C. Using these guidelines, thewashing temperature can be determined empirically, depending on thelevel of mismatch sought. Hybridization and wash conditions areexplained in Current Protocols in Molecular Biology (Ausubel, F. M. etal., eds., John Wiley & Sons, Inc., 1995, with supplemental updates) onpages 2.10.1 to 2.10.16, and 6.3.1 to 6.3.6.

High stringency conditions can employ hybridization at either (1) 1×SSC(10×SSC=3 M NaCl, 0.3 M Na3-citrate . . . 2H2O (88 g/liter), pH to 7.0with 1 M HCl), 1% SDS (sodium dodecyl sulfate), 0.1-2 mg/ml denaturedcalf thymus DNA at 65° C., (2) 1×SSC, 50% formamide, 1% SDS, 0.1-2 mg/mldenatured calf thymus DNA at 42° C., (3) 1% bovine serum albumin(fraction V), 1 mM Na2 . . . EDTA, 0.5 M NaHPO4 (pH 7.2) (1 M NaHPO4=134g Na2HPO4 . . . 7H2O, 4 ml 85% H3PO4 per liter), 7% SDS, 0.1-2 mg/mldenatured calf thymus DNA at 65° C., (4) 50% formamide, 5×SSC, 0.02 MTris-HCl (pH 7.6), 1×Denhardt's solution (100X=10 g Ficoll 400, 10 gpolyvinylpyrrolidone, 10 g bovine serum albumin (fraction V), water to500 ml), 10% dextran sulfate, 1% SDS, 0.1-2 mg/ml denatured calf thymusDNA at 42° C., (5) 5×SSC, 5×Denhardt's solution, 1% SDS, 100 μg/mldenatured calf thymus DNA at 65° C., or (6) 5×SSC, 5×Denhardt'ssolution, 50% formamide, 1% SDS, 100 μg/ml denatured calf thymus DNA at42° C., with high stringency washes of either (1) 0.3-0.1×SSC, 0.1% SDSat 65° C., or (2) 1 mM Na2EDTA, 40 mM NaHPO4 (pH 7.2), 1% SDS at 65° C.The above conditions are intended to be used for DNA-DNA hybrids of 50base pairs or longer. Where the hybrid is believed to be less than 18base pairs in length, the hybridization and wash temperatures should be5-10° C. below that of the calculated Tm of the hybrid, where Tm in °C.=(2× the number of A and T bases)+(4× the number of G and C bases).For hybrids believed to be about 18 to about 49 base pairs in length,the Tm in ° C.=(81.5° C.+16.6(log 10M)+0.41(% G+C)−0.61 (%formamide)−500/L), where “M” is the molarity of monovalent cations(e.g., Na+), and “L” is the length of the hybrid in base pairs.

Moderate stringency conditions can employ hybridization at either (1)4×SSC, (10×SSC=3 M NaCl, 0.3 M Na3-citrate. 2H2O (88 g/liter), pH to 7.0with 1 M HCl), 1% SDS (sodium dodecyl sulfate), 0.1-2 mg/ml denaturedcalf thymus DNA at 65° C., (2) 4×SSC, 50% formamide, 1% SDS, 0.1-2 mg/mldenatured calf thymus DNA at 42° C., (3) 1% bovine serum albumin(fraction V), 1 mM Na2 . . . EDTA, 0.5 M NaHPO4 (pH 7.2) (1 M NaHPO4=134g Na2HPO4 . . . 7H2O, 4 ml 85% H3PO4 per liter), 7% SDS, 0.1-2 mg/mldenatured calf thymus DNA at 65° C., (4) 50% formamide, 5×SSC, 0.02 MTris-HCl (pH 7.6), 1×Denhardt's solution (100X=10 g Ficoll 400, 10 gpolyvinylpyrrolidone, 10 g bovine serum albumin (fraction V), water to500 ml), 10% dextran sulfate, 1% SDS, 0.1-2 mg/ml denatured calf thymusDNA at 42° C., (5) 5×SSC, 5×Denhardt's solution, 1% SDS, 100 μg/mldenatured calf thymus DNA at 65° C., or (6) 5×SSC, 5×Denhardt'ssolution, 50% formamide, 1% SDS, 100 μg/ml denatured calf thymus DNA at42° C., with moderate stringency washes of 1×SSC, 0.1% SDS at 65° C. Theabove conditions are intended to be used for DNA-DNA hybrids of 50 basepairs or longer. Where the hybrid is believed to be less than 18 basepairs in length, the hybridization and wash temperatures should be 5-10°C. below that of the calculated Tm of the hybrid, where Tm in ° C.=(2×the number of A and T bases)+(4× the number of G and C bases). Forhybrids believed to be about 18 to about 49 base pairs in length, the Tmin ° C.=(81.5° C.+16.6(log 10M)+0.41(% G+C)−0.61 (% formamide)−500/L),where “M” is the molarity of monovalent cations (e.g., Na+), and “L” isthe length of the hybrid in base pairs.

Low stringency conditions can employ hybridization at either (1) 4×SSC,(10×SSC=3 M NaCl, 0.3 M Na3-citrate . . . 2H2O (88 g/liter), pH to 7.0with 1 M HCl), 1% SDS (sodium dodecyl sulfate), 0.1-2 mg/ml denaturedcalf thymus DNA at 50° C., (2) 6×SSC, 50% formamide, 1% SDS, 0.1-2 mg/mldenatured calf thymus DNA at 40° C., (3) 1% bovine serum albumin(fraction V), 1 mM Na2 . . . EDTA, 0.5 M NaHPO4 (pH 7.2) (1 M NaHPO4=134g Na2HPO4 . . . 7H2O, 4 ml 85% H3PO4 per liter), 7% SDS, 0.1-2 mg/mldenatured calf thymus DNA at 50° C., (4) 50% formamide, 5×SSC, 0.02 MTris-HCl (pH 7.6), 1×Denhardt's solution (100X=10 g Ficoll 400, 10 gpolyvinylpyrrolidone, 10 g bovine serum albumin (fraction V), water to500 ml), 10% dextran sulfate, 1% SDS, 0.1-2 mg/ml denatured calf thymusDNA at 40° C., (5) 5×SSC, 5×Denhardt's solution, 1% SDS, 100 μg/mldenatured calf thymus DNA at 50° C., or (6) 5×SSC, 5×Denhardt'ssolution, 50% formamide, 1% SDS, 100 μg/ml denatured calf thymus DNA at40° C., with low stringency washes of either 2×SSC, 0.1% SDS at 50° C.,or (2) 0.5% bovine serum albumin (fraction V), 1 mM Na2EDTA, 40 mMNaHPO4 (pH 7.2), 5% SDS. The above conditions are intended to be usedfor DNA-DNA hybrids of 50 base pairs or longer. Where the hybrid isbelieved to be less than 18 base pairs in length, the hybridization andwash temperatures should be 5-10° C. below that of the calculated Tm ofthe hybrid, where Tm in ° C.=(2× the number of A and T bases)+(4× thenumber of G and C bases). For hybrids believed to be about 18 to about49 base pairs in length, the Tm in ° C.=(81.5° C.+16.6(log 10M)+0.41(%G+C)−0.61 (% formamide)−500/L), where “M” is the molarity of monovalentcations (e.g., Na.+), and “L” is the length of the hybrid in base pairs.

Anti-LL-37 antibody can be produced recombinantly using a DNA sequencethat encodes the antigen, which has been inserted into an expressionvector and expressed in an appropriate host cell. DNA sequences encodingan anti-LL-37 antibody can, for example, be identified by assaying theantibody or fragment thereof against LL-37 and assessing binding and/oraffinity characteristics.

The invention also provides vectors, plasmids or viruses containing oneor more of the anti-LL-37 antibody nucleic acid molecules (e.g., havingthe sequence of SEQ ID NO:1, 3, or combinations thereof). Suitablevectors for use in eukaryotic and prokaryotic cells are known in the artand are commercially available or readily prepared by a skilled artisan.Additional vectors can also be found, for example, in Ausubel, F. M., etal., Current Protocols in Molecular Biology, (Current Protocol, 1994)and Sambrook et al., “Molecular Cloning: A Laboratory Manual,” 2nd ED.(1989).

Recombinant polypeptides containing portions and/or variants of a nativeantigen can be readily prepared from a DNA sequence encoding thepolypeptide using a variety of techniques well known to those ofordinary skill in the art. For example, supernatants from suitablehost/vector systems which secrete recombinant protein into culture mediacan be first concentrated using a commercially available filter.Following concentration, the concentrate can be applied to a suitablepurification matrix such as an affinity matrix or an ion exchange resin.Finally, one or more reverse phase HPLC steps can be employed to furtherpurify a recombinant protein.

Any of a variety of expression vectors known to those of ordinary skillin the art can be employed to express recombinant polypeptides of thisinvention. Expression can be achieved in any appropriate host cell thathas been transformed or transfected with an expression vector containinga DNA molecule that encodes a recombinant polypeptide. Suitable hostcells include prokaryotes, yeast and higher eukaryotic cells.Preferably, the host cells employed are E. coli, yeast or a mammaliancell line such as COS or CHO. The DNA sequences expressed in this mannercan encode naturally occurring antigens, portions of naturally occurringantigens, or other variants thereof.

Uses of plasmids, vectors or viruses containing the cloned anti-LL-37antibody sequences or fragments include one or more of the following;(1) generation of hybridization probes for detection and measuring levelof LL-37 in tissue; (2) generation of mRNA or protein in vitro or invivo; and (3) generation of transgenic non-human animals or recombinanthost cells.

In one embodiment, the present invention encompasses host cellstransformed with the plasmids, vectors or viruses described above.Nucleic acid molecules can be inserted into a construct which can,optionally, replicate and/or integrate into a recombinant host cell, byknown methods. The host cell can be a eukaryote or prokaryote andincludes, for example, yeast (such as Pichia pastorius or Saccharomycescerevisiae), bacteria (such as E. coli, or Bacillus subtilis), animalcells or tissue, insect Sf9 cells (such as baculoviruses infected SF9cells) or mammalian cells (somatic or embryonic cells, Human EmbryonicKidney (HEK) cells, Chinese hamster ovary cells, HeLa cells, human 293cells and monkey COS-7 cells). Host cells suitable in the presentinvention also include a mammalian cell, a bacterial cell, a yeast cell,an insect cell, and a plant cell.

The nucleic acid molecule can be incorporated or inserted into the hostcell by known methods. Examples of suitable methods of transfecting ortransforming cells include calcium phosphate precipitation,electroporation, microinjection, infection, lipofection and directuptake. “Transformation” or “transfection” as used herein refers to theacquisition of new or altered genetic features by incorporation ofadditional nucleic acids, e.g., DNA. “Expression” of the geneticinformation of a host cell is a term of art which refers to the directedtranscription of DNA to generate RNA which is translated into apolypeptide. Methods for preparing such recombinant host cells andincorporating nucleic acids are described in more detail in Sambrook etal., “Molecular Cloning: A Laboratory Manual,” Second Edition (1989) andAusubel, et al. “Current Protocols in Molecular Biology,” (1992), forexample.

The host cell is then maintained under suitable conditions forexpression and recovery of the anti-LL-37 antibody of the presentinvention. Generally, the cells are maintained in a suitable bufferand/or growth medium or nutrient source for growth of the cells andexpression of the gene product(s). The growth media are not critical tothe invention, are generally known in the art and include sources ofcarbon, nitrogen and sulfur. Examples include Luria broth, Superbroth,Dulbecco's Modified Eagles Media (DMEM), RPMI-1640, M199 and Grace'sinsect media. The growth media can contain a buffer, the selection ofwhich is not critical to the invention. The pH of the buffered Media canbe selected and is generally one tolerated by or optimal for growth forthe host cell.

The host cell is maintained under a suitable temperature and atmosphere.Alternatively, the host cell is aerobic and the host cell is maintainedunder atmospheric conditions or other suitable conditions for growth.The temperature should also be selected so that the host cell toleratesthe process and can be for example, between about 13-40 degrees Celsius.

EXEMPLIFICATION Example 1 Method of Making an Antibody

The following methods were used to significantly increase theantigenecity of most of peptides by a chemical modification.

A chemical called “methylglyoxal” can react to arginine (R), lysine (K)or cysteine (C) present in a peptide and increases the peptide'sabsorption by antigen presenting cells which express the specificreceptor (RAGE; the receptor for advanced glycation endproducts) formethylglyoxal-modified peptide 29.7% of total 37 amino acids are R or K.

The rule for this technology is that arginine (R), lysine (K) orcysteine (C) should be present in the peptide. Fortunately, LL-37 isabundant in both arginine (R) and lysine (K).

Using this method, at least three MAbs were generated and have bind athigh affinity to different epitopes present in LL-37. Since suchchemical modification has never published, none of commercial bio-labsor research scientists can develop high affinity antibodies to LL-37.

Methylglyoxal (MG; 40% aqueous solution) was purchased from Sigma.

Synthetic LL-37 (1 mg/ml; generated by a commercial service of Biomatikcorporation) was incubated with MG (1 mm) in phosphate buffered saline(PBS), pH 7.4, at 37° C. for 7 days, with adjustment of the pH to 7.4with sodium hydroxide solution (5 M) as required. To remove unbound freeMG, the resultant LL-37 solution incubated with MG was dialyzed againstPBS using a dialysis membrane (1,000 MW cut) for 3 days at 4 C. Afterfiltration of MG-modified LL-37 through 0.2 um pore size disc filter,the MG-modified LL-37 was stored in a −20C freezer.

The MG-modified LL-37 or intact non-treated LL-37 was immunized toBALB/c mice (8 week old male) in the Freund's Complete and IncompleteAdjuvants (FCA and FIA, respectively, from Difco). In order to make theemulsion of adjuvant, 1 vol of LL-37 (1 mg/ml) in PBS was mixed with 1vol of either FCA or FIA, followed by ultra-sonication of the mixture onice for 10-20 seconds that completes the formation of emulsion. Primaryimmunization of mice with LL-37 as well as secondary immunization wascarried out by s.c. injection of FCA emulsion and FIA emulsion (100 ugpeptide/200 ul emulsion/mouse, respectively) at the two week interval.Two weeks after the secondary immunization, booster immunization wasconducted by the i.v. injection of peptide in PBS (100 ug/peptide/100 ulPBS/mouse). Blood sera were sampled from the mice after the 3-5 daysfrom the booster immunization, and IgG antibody response to LL-37 wasmonitored using an ELISA. For the development of monoclonal antibody,animals were sacrificed 4 days after the booster immunization, andspleen cells isolated from the sacrificed mice was fused with NS-0 mousemyeloma cells using polyethylene glycol (PEG).

Using these Anti-LL-37 MAbs, the Following was Done:1) Develop ELISA to quantify the amount of LL-37 (none of thecommercially available antibody can be applicable for ELISA).2) Detect both pro-form (18 kD) and active form (4 kD) of LL-37 byWestern blot (All commercially available antibodies react to onlypro-form (18 kD) LL-37 or called CAP18)

Example 2 Sequencing of PCR Products

Two hybridoma cell lines that produce IgG antibody (IgG1 isotype) thatreact specifically to LL-37 were established. The two hybridoma celllines were termed as AL37-2 and AL37-7, respectively. Total RNA fromeach hybridoma cell line was extracted using a RNA-Bee solution (phenoland guanidine thiocyanate, TEL-TEST inc.) and chloroform. The amount ofisolated total RNAs were quantified by spectrometry at 260 and 280 nm.Five micrograms of each total RNA sample was used to perform afirst-strand cDNA synthesis using a reverse transcriptase (SuperScriptFirst-Strand Synthesis System, Invitrogen). The generated cDNA was thenused as a template DNA for the subsequent PCR using an Expandhigh-fidelity PCR system (Roche) with sense primer;5′-CCTGGGGCTTCAGTGAAG-3′ and antisense primer; 5′-GTGTCTTGCACAGTAATA-3′.Such primer set was designed to specifically bind to the frame worksequence present in the hyper variable region of mouse IgG1 heavy chain.The resulting PCR products were separated by electrophoresis on a 1.5%agarose gel and purified by gel extraction using Nucleospin (Clontech).The PCR products were subjected to DNA sequence using a commercialservice (Genwiz, Inc.). The sequences are shown in FIGS. 8 and 9.

Example 3 Induction of LL-37 Peptides

Induction of LL-37 and hBD2 mRNA Expression in Human Gingival EpithelialCells:

Both LL-37 and hBD2 mRNA expression in OBA9 cells are induced after a4-hour exposure to green tea polyphenol extract, Teaflan 90S (EGCG 50%,ECG 13%), and purified EGCG (95% pure, SIGMA) at concentrations of 0.1,1.0 and 10 ug/ml. EGCG increases the production of LL-37 and hBD2 at themRNA level in the human gingival epithelial cell line OBA9 (FIG. 1) aswell as the human lung carcinoma cell line A549.

In Vitro Induction of LL-37 Protein Expression in Human GingivalEpithelial Cells:

Using ELISA systems to detect LL-37 (Forsyth in-house) and hBD2(Peprotech), 33 natural compounds (20 ug/ml) are examined for theirability to induce LL-37 and hBD2 from OBA9 cells. Although thesensitivity of the hBD2 ELISA is higher than that of LL-37 ELISA (10pg/ml vs 1 ng/ml, respectively), the induction of LL-37 is moreprominent than hBD2 (FIG. 2 A&B, LL-37; C&D, hBD2). Trihydroxybenzoatederivatives and control mitogenic agents, PMA and Poly IC, inducedLL-37, whereas Trihydroxybenzoate derivatives, PMA and Poly IC haveminimal induction activities. Bactericidal effects of LL-37 and hBD2are, in general, potent. Therefore, although hBD2 induction wassignificantly elevated compared to the non-stimulated control, theconcentration of expressed hBD2 appeared insufficient to kill bacteria.

The Trihydroxybenzoate Derivatives of the Invention and rosemarinicacid, courmarin, catechin gallate, epicatechin gallate,epigallocatechin, and tannic acid are tested to induce LL-37 from OBA9cells in the absence of EGCG. Rosemarinic acid and courmarin, which lack3,4,5-trihydroxy benzoate, showed little or no induction of LL-37expression by OBA9 cells. Therefore, gallic acid appears to be morepotent than 3,4,-dihydroxy benzoate in the induction of LL-37 expressionby cultured gingival epithelial cells.

Culture Supernatant of EGCG-Treated Human Gingival Epithelial Cells:

The culture supernatant isolate from OBA9 is treated with 1) EGCG, 2)catechin, 3) 2,4-dihydroxy benzoic acid, and 4) 3,4,5-tri-hydroxybenzoic acid (20 ug/ml, respectively) and examined for theirbactericidal effects on Actinobacillus actinomycetemcomitans Y4 (Aa Y4).The culture supernatant of EGCG- and 3,4,5-tri-hydroxy benzoicacid-treated OBA9 cells show more bactericidal effects than controlnon-treated OBA9 supernatant or supernatant of OBA9 treated withcatechin or with 2,4-dihydroxy benzoic acid. Therefore,3,4,5-tri-hydroxy benzoic acid and EGCG which contains gallic acid bothappear to have higher antimicrobial effects than the compounds which donot have the structural isomers of gallic acid.

In Vivo Induction of LL-37 mRNA and Protein Expression by Human CheekEpithelial Cells:

Cheek epithelium is utilized for monitoring LL-37 mRNA and proteinexpression to determine if Trihydroxybenzoate Derivatives of theInvention can induce LL-37 in vivo. Total RNA is extracted from cheekepithelial cells after application of mouthwash with EGCG (40 ug/ml),and LL-37 mRNA expression by the cheek epithelial cells is determined byRT-PCR. mRNA for LL-37 is up-regulated after only 5 minutes fromapplication of the mouthwash with EGCG, whereas the internal controlβ-actin is expressed constitutively. The isolated cheek cells are alsosubjected to Western blot analysis for protein expression of LL-37 bycheek epithelium using anti-LL-37 monoclonal antibody. The expression ofboth the proform (18 kD, also termed as hCAP18) and secreted form (4.5kD) of LL-37 (12) in cheek epithelial cells is induced byTrihydroxybenzoate Derivatives of the Invention (e.g., EGCG (95% SIGMA),or by EGCG (90% Teavigo)). The immunohistochemical analysis for LL-37expression stained with anti-LL-37 monoclonal antibody also demonstratethe increased expression of LL-37 in the cheek epithelial cells after exvivo stimulation.

Tri-Hydroxy Benzoic Acid:

In order to investigate the structure-function relationship underlyingEGCG-mediated induction of LL-37 from human gingival epithelial cellline OBA9, different isomers of benzoic acids are examined for theireffects on the LL-37 expression by OBA9 cells. Each isomer of benzoicacids is coupled to NHS-activated Sepharose gel (Pierce) using across-linker, 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC,Pierce). The isomers of benzoic acids include 1) 2,6-di-hydroxy benzoicacid, 2) 2,3,4-tri-hydroxy benzoic acids, 3) 3,4,5-tri-hydroxy benzoicacids, and 4) 2,4,6-tri-hydroxy benzoic acids. Only 2,3,4-tri-hydroxybenzoic acids and 3,4,5-tri-hydroxy benzoic acids, but not2,6-di-hydroxy benzoic acids or 2,4,6-tri-hydroxy benzoic acids, showthe induction of LL-37 expression by OBA9 cells.

The teachings of related Application No. (not yet assigned, attorneyDocket no: 0024.0012-002), filed Apr. 16, 2010, entitled “AntibacterialCompositions” by Toshihisa Kawai, et al. are incorporated herein byreference in this entirety.

The relevant teachings of all the references, patents and/or patentapplications cited herein are incorporated herein by reference in theirentirety.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A method of preparing an immunizing antigen to be used in making anantibody to a native antigen, the method comprises the steps of: a.selecting a native antigen having an amino acid sequence with a highcontent of arginine, lysine, cysteine or a combination thereof; and b.contacting the antigen with methylglyoxal in an amount sufficient tochemically modify the antigen to thereby obtain a chemically modifiedantigen; wherein the chemically modified antigen is prepared for use inmaking an antibody specific to the native antigen.
 2. The method ofclaim 1, further comprising selecting an amino acid sequence having acontent of arginine, lysine, cysteine or a combination thereof in arange between about 1% and about 30%.
 3. The method of claim 1, furthercomprising contacting the antigen with an amount of methylglyoxal in arange between about 1 nM and about 1 mM.
 4. The method of claim 1,further comprising immunizing an animal with the chemically modifiedantigen.
 5. A method of making an antibody to a native antigen, themethod comprises the steps of: a. selecting a native antigen having anamino acid sequence with a high content of arginine, lysine, cysteine ora combination thereof; b. contacting the antigen with methylglyoxal inan amount sufficient to chemically modify the antigen to thereby obtaina chemically modified antigen; and c. injecting the chemically modifiedantigen into an animal in an amount sufficient to illicit an immuneresponse by the animal, wherein the immune response includes theproduction of one or more antibody secreting cells that is specific tothe native antigen; wherein the antibody to the native antigen is made.6. The method of claim 5, further comprising contacting the antibodysecreting cell with a myeloma cell under conditions suitable for fusionthereof to thereby obtain a hybridoma that secretes an antibody specificto the native antigen.
 7. The method of claim 6, further comprisingselecting for hybridomas made during fusion.
 8. The method of claim 7,further comprising isolating the hybridoma that secretes an antibodyspecific to the native antigen.
 9. An antibody made from the methods ofclaim
 5. 10. An isolated polypeptide molecule comprises an amino acidsequence selected from the group consisting of: a. an amino acidsequence encoded by a nucleic acid molecule having a sequence of SEQ IDNO: 1, 3, or combination thereof; b. an amino acid sequence encoded by acomplement of SEQ ID NO: 1, 3, or combination thereof; c. an amino acidsequence encoded by a nucleic acid molecule that hybridizes to SEQ IDNO: 1, 3, or combination thereof; and d. an amino acid sequence setforth in SEQ ID NO: 2, 4, or combination thereof.
 11. An isolatednucleic acid molecule having a nucleic acid sequence selected from thegroup consisting of: a. a nucleic acid sequence set forth in SEQ ID NO:1, 3, or combination thereof; b. a nucleic acid sequence that is acomplement of SEQ ID NO: 1, 3, or combination thereof; c. a nucleic acidsequence that hybridizes to SEQ ID NO:1, 3, or combination thereof; andd. a nucleic acid sequence that encodes SEQ ID NO: 2, 4, or combinationthereof.
 12. The isolated nucleic acid molecule of claim 11, furthercomprising a nucleic acid sequence that encodes a detectable label. 13.The isolated nucleic acid molecule of claim 12, wherein the detectablelabel is luciferase.
 14. A vector or plasmid that comprises the nucleicacid molecule of claim
 11. 15. A host cell transformed with the nucleicacid sequence of claim
 11. 16. A composition that comprises thepolypeptide sequence of claim 10 and a physiologically acceptablecarrier.
 17. An antibody specific to LL-37, wherein the antibodycomprises a variable heavy chain having an amino acid sequence of claim10.
 18. The antibody specific to LL-37 of claim 16, further comprising adetectable label.
 19. A method for measuring the presence, absence oramount of LL-37 in a sample, wherein the method comprises: a. contactingthe sample with an antibody that binds to LL-37 sufficient to allowformation of a complex between the sample and the antibody, to therebyform an antigen-antibody complex; and b. assessing the presence, absenceor amount of the antigen-antibody complex.
 20. The method of claim 19,further comprising comparing the amount of the antigen-antibody complexto a control.
 21. The method of claim 19, wherein said antibody isdetectably labeled.
 22. The method of claim 19, wherein the methodfurther includes contacting the sample with a second antibody specificto LL-37 or said antigen-antibody complex.
 23. The method of claim 19,wherein the antibody that binds to LL-37 has an amino acid sequence setforth in SEQ ID NO: 1,3, or combination thereof.
 24. The method of claim19, wherein the anti-microbial peptide or the antibody is bound to asolid support.
 25. A method for assessing a compound for stimulation ofLL-37 in a sample, wherein the method comprises: a. contacting thesample with a compound to be tested to allow stimulation of LL-37; b.contacting the sample with an antibody that binds to LL-37 sufficient toallow formation of a complex between the sample and the antibody, tothereby form an antigen-antibody complex; and c. assessing the presence,absence or amount of the antigen-antibody complex; wherein the presenceor an increased level of the LL-37, as compared to a control, indicatesthat the compound stimulates production of the LL-37, and the absence ordecreased level of LL-37, as compared to a control, indicates that thecompound does not stimulate of the production of the LL-37.
 26. A methodfor assessing a compound for stimulation of LL-37 in a sample, themethod comprises: a. contacting the sample with the compound to betested sufficiently to allow stimulation of LL-37; b. contacting thesample with at least two oligonucleotide primers in a polymerase chainreaction, wherein at least one of the oligonucleotide primers isspecific for the nucleic acid sequence of LL-37, sufficiently to allowamplification of the primers; and c. detecting in the sample theamplified nucleic acid sequence; wherein the presence the amplifiednucleic acid sequence indicates that the compound stimulates productionof the LL-37, and the absence of the amplified nucleic acid sequenceindicates that the compound does not stimulate of the production of theLL-37.
 27. The method of claim 26, wherein at least one of theoligonucleotide primers comprises at least about 10 contiguous bases.28. A method for assessing a compound for stimulation of LL-37 in asample, the method comprises: a. contacting the sample with the compoundto be tested sufficiently to allow stimulation of LL-37; b. contactingthe sample with one or more oligonucleotide probes specific for anisolated nucleic acid molecule of LL-37 under high stringencyconditions, sufficiently to allow hybridization between the sample andthe probe; and c. detecting the nucleic acid molecule that hybridizes tothe oligonucleotide probe in the sample; wherein the presence thehybridization indicates that the compound stimulates production of theLL-37, and the absence of hybridization indicates that the compound doesnot stimulate of the production of the LL-37.